The broad definition of pain includes neural processes that take place to identify internal or external environmental influences that pose a risk for tissues of the body. Neural systems first calculate the magnitude of the risk. Depending on this magnitude, the systems will generate the driving force for a battery of behaviors aimed at minimizing the risk of tissue damage. For localized high intensity stimuli (such as when a hot stove is touched) speed of the response is of highest priority. There is no time for an in-depth analysis of all aspects of the threat, and information about location and intensity of the stimulus dominate the process that leads to a stereotypical but rapid first response (reflex withdrawal). For lower intensity or diffuse stimuli, or for stimuli where reflexes have failed to bring the desired result, higher-order neural circuitry is recruited for an in-depth analysis of the threat and generation of a response adapted to the specific situation. The signal that arrives from the site of tissue insult will be temporally and spatially integrated because the risk (and thus urgency of response) is a function of intensity x duration x spatial extent of the stimulus. The experiential and behavioral result of this computation may be subject to modification to better adapt the response to situational factors that are important for the survival of the animal as a whole. For instance, surviving a fight or flight situation takes precedence over protecting an injured paw, and thus pain from the paw will be suppressed until the animal has reached a safer environment.
The clinical definition of pain relates to the net result of many steps of neural processing. Typically, the definition is limited to the input side of the phenomenon of pain, namely the conscious experiential aspects. The output stage (escape, vocalization, verbal response) is often not included in the narrow definition of pain, but merely considered a means to infer the magnitude of the sensory or affective experience. Conscious (clinically most relevant) aspects of pain are the result of many more steps of neural processing than simple withdrawal reflexes. Clinical pain may be generated under conditions that do not lead to activation or facilitation of reflexes. Furthermore, suppression of protective reflexes is not an objective of pain medicine and may not be in the interest of the patient at all. The goal is reduction of the conscious unpleasant, often persistent experience of pain. Animal models, to be clinically relevant, must be predictors of this conscious pain experience.
Pain is an experience that cannot be measured directly, either in humans, or in animals, but must be inferred from behaviors. The available repertoire of behaviors that consistently reveal pain includes verbalizations in humans and complex motor sequences that eliminate nociceptive stimulation (escape responses) in humans and other animals. A variety of other behaviors suggest the presence of pain but can be elicited by stimuli or situations that are not necessarily aversive or involve responses that do not require a conscious perception of pain. Pain tests for non-human animals have been reviewed extensively in the literature (Vierck, C. J., B. Y. Cooper, Advances in Pain Research and Therapy [1984], pp. 305-322; Chapman, C. R et al., Pain [ 1985] 22:1-31; Dubner, R. Textbook of Pain [ 1989], pp. 247-256; Franklin, K. B. J and F. V. Abbott, Neuromethods, Psychopharmacology [1989] 13:145-215; Vierck, C. J. et al., Issues in Pain Measurement [1989] pp. 93-115), and they can be classified according to two main criteria: (I) type of stimulus applied; and (2) type of response measured.
Some methods for evaluating phasic responses to nociceptive stimulation involve electrical stimulation, because it can be turned on and off instantly, making it easy for an animal to learn the temporal relationship between an escape response and elimination of an aversive sensation. Although electrical stimulation has been criticized because skin receptors are bypassed, and synchronous afferent firing patterns are generated (Dubner, R., 1989), it is possible to elicit natural sensations of predictable quality when electrode tissue coupling is tightly controlled (Vierck, C. J. et al., Animal Pain Perception and Alleviation; American Physiological Society [1983a] pp. 117-132; Vierck, C. J. et al., 1989; Vierck, C. J. et al., Somatosens Mot Res [1995] 12:163-174). However, control over current density and stimulus location can be achieved only by restraining the subjects, and animals will tolerate restraint only after lengthy adaptation and training periods. Restraint without proper adaptation leads to high levels of stress and anxietyxe2x80x94factors that are known to have modulatory effects on pain sensitivity (Amir, S. and Z. Amit, Life Sci [1978 ] 23:1143-1151; Bhattacharya, S. K. et al., Eur J Pharmacol [1978] 50:83-85; Basbaum, A. I. and H. L. Fields, Annu Rev Neurosci [1984] 7:309-338; Franklin, K. B. J. and F. V. Abbott, 1989; Maier, S. F. et al., APS J [1992] 1:191-198; Tokuyama, S. et al., Jpn J Pharmacol [1993] 61:237-242; Caceres, C. and J. W. Burns, Pain [1997] 69:237-244). Therefore, nociceptive tests that require restraint or extensive handling, which have an effect on pain processing, may produce contaminated results.
Thermal stimulation has been used previously for nociceptive tests (Dubner, R., 1989). Contact thermal stimulation provides the basis for the hotplate test (Woolfe, G and A. D. Macdonald, J Pharmacol Exp Ther [1944] 80:300-307), and extensive use of contact heat in psychophysical and neurophysiological studies has established the range of temperatures that produces heat nociception. Radiant heat is used in the tailflick test (D""Amour, F. E. and D. Smith, J Pharmacol Exp Ther [1941] 72:74-79) and the Hargreaves hindlimb-withdrawal test (Hargreaves, K et al., Pain [1988] 32:77-88). The absence of a concurrent mechanical stimulus is thought to be an advantage of radiant heat, but it is difficult to control and assess skin temperature. Observations of hindlimb withdrawal and/or guarding behavior have also been utilized to evaluate thresholds for reactivity to mechanical stimulation (Chaplan, S. R. et al. J Neurosci Methods [1994] 53:55-63) or the frequency of responsivity to chemical stimulation (Dubuisson, D and S. G. Dennis, Pain [1977] 4:161-174). A present difficulty with mechanical tests is that characteristics of von Frey filaments (e.g. combinations of diameter and force) which produce mechanical nociception have not been determined. Chemical stimuli can be varied in concentration, volume and method of application (injection or surface application), but it is difficult to characterize the effects of these agents on peripheral tissues, receptors and afferents. These different methods of nociceptive testing elicit responses that can be modulated differentially by a variety of treatments (Willer, J. C. et al. Brain Res [1979] 179:61-68; McGrath, P. A. et al., Pain [1981] 10:1-17; Vierck, C. J. et al., Progress in Psychobiology and Physiological Psychology [ 1983] pp. 113-165; Sandkuhler, J. and G. F. Gebhart, Brain Res [1984] 305:67-76; Dubner, R., 1989), and it is often concluded that the method of stimulation is the determinant factor, without consideration of other aspects of the testing method and response measurement.
An important consideration in evaluation of nociceptive tests is the central circuitry that is interposed between the input and output stages. For example, the tail flick and paw withdrawal responses can be elicited in spinal animals (Franklin, K. B. J. and F. V. Abbott, 1989) and therefore can represent segmental spinal reflexes. Pawlicking in the hot-plate test (Woolfe, G. and A. D. Macdonald, 1944; Eddy, N. B. et al., J Pharmacol Exp Ther [1950] 98:121-137; Chapman, C. R et al., 1985) and vocalization (Carroll, M. N and R. K. S. Lim, Arch Int Pharmacodyn [1960] 125:383-403) can be elicited in chronic decerebrate rats (Woolf, C. J., Pain [1984] 18:325-343; Berridge, K. C., Behav Brain Res [1989] 33:241-253; Matthies, B. K. and K. B. Franklin, Pain [1992] 51:199-206) and can be modulated differentially from responses to the same stimulus that originate at higher levels of the neuraxis (Sandkuhler, J. and G. F. Gebhart, 1984; Cooper, B. Y. and C. J. Vierck, Pain [1986] 26:393-407; Dubner, R., 1989). Therefore, it is important to distinguish innate responses that can be segmental (spinal) reflexes or long-loop (spino-bulbospinal) reflexes from operant responses that necessarily employ complex learned motor actions (involving the cerebrum).
Animal models of pain are most useful when they are good predictors of the effect of disease states or therapeutic interventions on human clinical pain. The clinically most relevant consequence of nociceptive stimuli is the conscious experience of pain and suffering that the stimuli may elicit. Assays based upon short or long-loop reflexes (such as the tail-flick test, paw withdrawal test, or hotplate test) provide little or no insight into what goes on at the conscious level. Reflex tests and the few available assays of conscious responses to painful stimuli, such as the foot shock escape test, rely mostly on fast-conducting pain pathways. However, it is known that slow-conducting nociceptive systems are the major contributors to the conscious experience of clinical pain and they are primarily affected by powerful pain killers such as morphine.
Shuttle-box paradigms, using the operant response measure of learned escape have been popular models of conscious aspects of pain (Warner, L. H., J Genetic Psychol [1932] 41:57-89; Bohus, B. and D. Wied, J Comp Physiol Psychol [1967] 64:26-29; Randall, P. K. and D. C. Riccio, J Comp Physiol Psychol [1969] 69:550-553; Cleary, A. Instrumentation for Psychology [1977] pp. 1-319). These methods are easy to implement, because the subjects are unrestrained. Electrical stimulation has been used in shuttle box paradigms because it can be regulated in intensity and switched between chambers. However, it is problematic in these situations, because movement of the animals across a grid floor switches polarities and varies current densities.
A shuttle-box test was developed which uses thermal nociceptive stimulation (hot or cold), as opposed to electrical stimulation (Mauderli, A. P. et al., J Neurosci Methods [2000] 97:19-29). The thermal stimulus is transmitted from a thermal plate on the floor of a compartment to the paws of the freely moving animals. However, through training, the animals learn that they can escape the stimulus by moving from the heated compartment, which is kept dark, onto a non-heated platform within an enclosure, which is suspended within the heated compartment. Thus, the enclosed platform represents the destination for escape from the thermal plate. However, to discourage avoidance behavior, the escape platform is made less attractive by brightly illuminating it and imposing a degree of tilt toward the dark compartment. The thermal plate is kept constantly heated by internal water circulation and a new trial is started by swinging the enclosure into a vertical position, thereby ejecting the animal onto the thermal plate.
Tests of nociception are most often used to evaluate pharmacological, disease, or surgical effects on pain. However, these effectors may alter the measured behaviors through mechanisms other than pain. Morphine, for instance, may make the animal sluggish in response to any stimulus, including non-painful stimuli. Therefore, it is necessary to pair any pain test with a valid control test for non-pain-related effects, such as attentional, motivational and motor effects (Dubner, R., 1989). To be valid, treatment effects on escape should be compared with effects on a control task that involves a comparable motivation (escape) and the same motor response (e.g. stepping on a platform) as the nociceptive test. The rotarod test (Dunham, N. W. and T. S. Miya, J Am Pharm Assoc [1957] 46:208-209; Kinnard Jr., W. J. and C. J. Carr, J Pharmacol Exp Ther [1957] 121:354-361) for instance, cannot be considered an adequate control for a reflex-based test or an operant shuttle-box assay of nociception, because the motor tasks and motivations differ considerably for these tests.
A control test for the thermal escape test was developed (Mauderli, A. P. et al., 2000), which measures latencies for escape from a bright light (controlling for generalized effects on aversion). In this arrangement, a two-chambered box that uses only bright light as the behavioral driving force is utilized for motor and general motivational effects for the thermal pain test. which is conducted in a separate apparatus. Therefore, the motor task in the control test is similar to that required by the thermal escape test, in that both involve escape into another compartment and use of a bright light. However, there are differences in the apparatuses used in the control test and thermal escape test that may limit their effectiveness. The differences in design between the two apparatuses requires that each animal be trained on both tasks and be able to distinguish between them. In addition, responses in the thermal escape test are always directed toward the same compartment (a one-way shuttle test), but the control test is a two-way test, because the aversive light can occur in either of the two chambers. A one-way shuttle test carries the risk that the animal can learn to associate between xe2x80x9ccomfortxe2x80x9d and a specific compartment or location. In addition, for experimental treatments that influence memory, it can be a drawback if the difficulty level of the two tests is not the same.
It is evident that the behavioral testing devices currently available may be of limited use as research tools with respect to nociception. Accordingly, there remains a need for a device which is capable of testing pain based upon a conscious response, permits assessment of slowly-conducting pain systems, avoids restraint stress, minimizes animal handling artifacts, and is matched with a valid behavioral and motor control test.
The subject invention concerns an apparatus for testing pain sensitivity exhibited by an animal. The apparatus can be used, for example, to evaluate the effect of a disease state, drug, or other intervention, on pain sensitivity. The apparatus is designed to measure a conscious escape response to a painful stimulus in test animals, such as rodents. The apparatus of the subject invention provides an inclusive operant pain test and a matching motor control test. The subject invention also pertains to methods for testing the pain sensitivity exhibited by a test animal, using the subject apparatus.
In a preferred embodiment, the apparatus of the subject invention includes two chambers, a first chamber and a second chamber, which are connected by a passageway. The passageway is of sufficient size to permit the test animal to pass between the chambers. Preferably, the passageway is designed such that the test animal can pass through the passageway even if the test animal is tethered to infusion cannula(s), cable(s), or other diagnostic and/or delivery device(s). The subject invention also includes single chamber embodiments.
Optionally, wireless telemetry can be utilized to transmit and receive diagnostic information (e.g., biological information) concerning the test animal. Wireless telemetry systems include, for example, radio-electric transmission, optical transmission, ultrasound transmission, or other transmission technologies that do not rely on a continuous wire, lead, or cable connection between the test animal and any external equipment.
The apparatus also includes means for presenting a painful stimulus to the test animal when the test animal is within the first chamber and a means for presenting a painful stimulus to the test animal when the test animal is within the second chamber. The means for presenting a painful stimulus presents a stimulus that is aversive and at least potentially painful to the test animal within the chamber where the means for presenting the painful stimulus is activated and in which the painful stimulus is produced. Preferably, the painful stimulus can motivate the test animal to exhibit an escape response, such as exiting the chamber in which the painful stimulus is presented.
Preferably, the means for presenting a painful stimulus to the test animal when the test animal is within the first chamber produces a painful condition (environment) within the first chamber. Preferably, the means for presenting a painful stimulus to the test animal when the test animal is within the second chamber produces a painful condition (environment) within the second chamber. Each means for presenting a painful stimulus can be independently activated and deactivated. Each painful stimulus can be independently and rapidly presented and independently and rapidly removed.
The apparatus further includes means for presenting an aversive, non-painful stimulus to the test animal when the test animal is within the first chamber and a means for presenting an aversive, non-painful stimulus to the test animal when the test animal is within the second chamber. The means for presenting an aversive, non-painful stimulus when the test animal is within the first and second chamber can each motivate the test animal to exhibit an escape response, such as exiting the chamber in which the aversive, non-painful stimulus is presented to the test animal. Preferably, the means for presenting an aversive, non-painful stimulus to the test animal when the test animal is within the first chamber is a means for producing an aversive, non-painful condition (environment) within the first chamber. Preferably, the means for presenting an aversive, non-painful stimulus to the test animal when the test animal is within the second chamber is a means for producing an aversive, non-painful condition (environment) within the second chamber. The means for presenting an aversive, non-painful stimulus can be independently activated and deactivated. Each aversive, non-painful stimulus can be independently and rapidly presented and independently and rapidly removed. Therefore, by presenting a painful stimulus and/or an aversive, non-painful stimulus to the test animal, the test animal can be motivated to exhibit an escape response, such as exiting the chamber it is occupying and moving through the passageway into another chamber.
Preferably, each means for producing an aversive, non-painful condition is one or more light sources for lighting the interior of the respective chamber, thereby creating a lit environment. Preferably, each means for producing a painful condition is a means for independently heating and cooling the respective chamber floor.
Optionally, the apparatus can include a means for exhibiting an escape response (other than the test animal""s movement between chambers). The escape response exhibiting means can be, for example, a lever, button, switch, or other actuating mechanism that, when activated by the test animal, terminates or lessens the magnitude of the aversive, non-painful stimulus or the painful stimulus. Thus, the test animal""s activation of the escape response exhibiting means is the test animal""s escape response (escape behavior). In addition, the apparatus can include a means for implementing an appetitive stimulus, which can likewise be activated by a lever, button, switch, or other actuating mechanism.
Optionally, the apparatus can include a tether system or wireless system to transmit signals to the test animal (e.g., electrical stimuli, chemical stimuli, pharmacological stimuli, optical signals, taste signals, olfactory signals, thermal signals). The signal sent to the test animal via tether or wireless link can be painful, aversive and non-painful, appetitive, addictive, or it can have a modulatory effect on the processing of stimuli delivered by other means (e.g., heating and cooling of the chamber floor). The signal sent to the test animal can be designed to generate anxiety, depression, or other emotional states, some of which are known to have a profound effect on pain processing. The trained test animal can choose when to initiate or terminate the stimulus (painful, aversive and non-painful, appetitive, addictive) by moving from one chamber into the other, or by activating the escape response exhibiting.
The apparatus can also include means for sensing the presence of the test animal within the first chamber and a means for sensing the presence of the test animal within the second chamber. The apparatus can be automated and used in conjunction with computer software for control of experiment conditions, response measurements, and data analysis.
The subject invention also concerns methods for using the subject apparatus to conduct escape latency tests, place preference tests, and control tests for each.